The present invention relates, in general, to biosensors and, in particular, to a glucose biosensor comprising a genetically engineered Glucose Binding Protein.
Biosensors couple highly specific biomolecular ligand binding events to changes in physical signals, thereby providing analytical tools that can measure the presence of single molecular species in complex mixtures. (Hall, Biosensors, Prentice-Hall: Englewood Cliffs (1991)). Most biosensors are naturally occurring macromolecules, such as enzymes or antibodies, which provide the desired analyte specificity, but often are not well suited to simple signal transduction mechanisms. (Griffiths et al, Tr. Biotech. 11:122-130 (1993)). One solution to this problem is to use protein engineering techniques to integrate signal transduction functions directly into proteins, adapting them to straightforward detection technologies, rather than developing instrumentation specific to the properties of a particular protein (Adams et al, Nature 39:694-697 (1991); Braha et al, Chem. Biol. 4:497-505 (1997); Brennan et al, Proc. Natl. Acad. Sci. U.S.A. 92:5783-5787 (1995); Brune et al, Biochemistry 33:8262-8271 (1994); Cornell et al, Nature 387:580-583 (1997); Gilardi et al, Anal. Chem. 66:3840-3847 (1994); Godwin et al, J. Am. Chem. Soc. 118:6514-6515 (1996); Marvin et al, Proc. Natl. Acad. Sci. U.S.A. 94:4366-4371 (1997); Post et al, J. Biol. Chem. 269:12880-12887 (1994); Romoser, J. Biol. Chem. 272:13270-13274 (1997); Stewart et al, J. Am. Chem. Soc. 116:415-416 (1994); Thompson et al, J. Biomed. Op. 1:131-137 (1996); Walkup et al, J. Am. Chem. Soc. 119:5445-5450 (1997)). A simple approach to building such integrated signal transducers is to exploit optical detection strategies based on changes in fluorescent reporter groups which respond to ligand binding (Guiliano et al, Annu. Rev. Biophys. Biomolec. Struct. 24:405-434 (1995); Czarnik, Chem. Biol. 2:432-438 (1995)). Fluorophores can be site-specifically introduced into a protein by using total synthesis, semi synthesis, or gene fusions. In this way pairs of fluorophores can be arranged for detection of binding by fluorescence energy transfer, or a single, environmentally-sensitive fluorophore can be positioned to respond to conformational changes accompanying binding events. (See references cited above.)
Ideally, the structural relationship between ligand binding site and reporter group is such that each can be manipulated independently, allowing a modular approach to the optimization of the properties of the binding site or the fluorophore. (Marvin et al, Proc. Natl. Acad. Sci. U.S.A. 94:4366-4371 (1997); Walkup et al, J. Am. Chem. Soc. 119:3443-3450 (1997); Cheng et al, J. Am. Chem. Soc. 118:11349-11356 (1996); Ippolito et al, Proc. Natl. Acad. Sci. U.S.A. 92:5017-5021 (1995); Elbaum et al, J. Am. Chem. Soc. 118:8381-8387 (1996)). One way to achieve such modularity is to spatially separate the two sites to minimize steric interference between them. Spatial separation of the reporter group and the binding site requires that the behavior of the fluorophore remain coupled to the degree of occupancy of the ligand binding site via an allosteric linkage mechanismn. Recently, it has been shown that it is possible to engineer such integrated fluorescent allosteric signal transducer (FAST) functions in the Maltose Binding Protein (MBP) of E. coli by taking advantage of the large conformational changes that occur upon ligand binding in this protein, using a structure-based rational design approach (Marvin et al, Proc. Natl. Acad. Sci. U.S.A. 94:4366-4371 (1997)).
The present invention relates, in one embodiment, to a Glucose/Galactose Binding Protein with engineered FAST functions and to a new class of fluorescent glucose sensors with applications in the food industry (Suleiman et al, In:Biosensor Design and Application, Matthewson and Finley, Eds. American Chemical Society, Washington, D.C., Vol. 511 (1992)), and clinical chemistry (Wilkins et al, Med. Eng. Phys. 18:273-278 (1996); Pickup, Tr. Biotech. 11:285-291 (1993); Meyerhoff et al, Endricon 6:51-58 (1996)).
The present invention relates to a glucose biosensor comprising a genetically engineered Glucose Binding Protein (GBP). In a specific embodiment, the invention relates to a GBP engineered to include mutations that allow site specific introduction of environmentally sensitive reporter groups. The signal of these prosthetic groups changes linearly with the degree of glucose binding. Thus, the glucose sensor of the invention can be used, for example, for detection of glucose in blood or industrial fermentation processes.